This proposal is now in its fourth cycle of competitive funding and focuses on Interleukin 15 (IL-15) and its role in human natural killer (NK) cell development, NK cell effector function and cancer. Our specific aims remain relatively unchanged: 1) To further identify and characterize progenitors and precursors that differentiate into human NK cells; 2) To better understand the role of IL-15 in regulating human NK cell effector functions. IL-15 induced differentiation and activation of NK cells are, like other biologc systems balanced by negative regulators, and those negative regulators are in turn exploited by tumors to turn off and evade the NK cell's ability to effectively survey against malignant transformation. Thus, our overall goal in this proposal is to gain a comprehensive understanding of the cellular and molecular components that constitute these inhibitory pathways in order to best understand how they work and how they can be modified to enhance NK cell development and function in the face of tumor challenge. In particular, in Aim 1 we will further investigate th innate cellular heterogeneity within secondary lymphoid tissues where we previously discovered and characterized human NK cell developmental intermediates. Over the past five years, an abundance of additional innate lymphoid cells (ILC) have been discovered and characterized, lending some controversy as to the origin of human NK cells. We hypothesize that the transcription factor aryl hydrocarbon receptor (AHR) acts to prevent human NK cell development from an IL-22 secreting ILC, and does so via the upregulation of miR-29b. Indeed, certain tumors have been shown to secrete AHR agonists that we postulate inhibit NK cell development. We propose a series of experiments to test our hypothesis, which if validated, will provide a new target for combating immune evasion. In Aim 2 we have preliminary data that suggest the activated Axl/Mer/Tyro3 receptor tyrosine kinase (RTK) pathway serves to negatively regulate IL-15-induced NK cell activation. Further, we have shown that the ligand to this RTK family, Gas6, is expressed on human acute myeloid leukemia cells and confers a poor prognosis. Thus, we hypothesize that this family of RTKs plays an important role in the negative regulation of NK cell effector functions and thus has the potential to be targeted for immune checkpoint inhibition in order to enhance NK cell-mediated immune therapy against cancer.